1. Field of the Invention
The present invention relates to improvements in flow cytometers of the type commonly used to differentiate small particles, e.g., various types of blood cells, in a liquid suspension. More particularly, this invention relates to improvements in flow cytometers of the type that use plane-polarized radiation, e.g., that emitted from laser diodes, to irradiate individual particles passing through an optical flow cell in order to detect the light-scattering characteristics of such particles and thereby characterize each particle as being a member of a particular class or type.
2. The Prior Art
Flow cytometers are commonly used to differentiate individual small particles of different types in a particle suspension on the basis of the light-scattering and/or fluorescence characteristics of each particle. Such instruments generally include an optically-transparent flow cell having a particle-interrogation zone through which particles from the sample are made to pass in single file; a laser light source for irradiating such particles, one-at-a-time, as they pass through the particle-interrogation zone; and a plurality of optical detectors that are strategically located about the flow cell to receive both scattered radiation from the irradiated particles, and fluorescence radiation emitted by fluorochromes that have been previously attached to certain particles in a class or of a type of interest. Typically, the photodetectors are positioned to detect both forwardly-scattered radiation within angular ranges determined by the geometry of the light-sensitive elements of the photodetector, and side-scattered radiation that is scattered in direction substantially perpendicular to the directions of the irradiating laser beam and of the particle path. The respective outputs of the photodetectors are then processed in a known manner to identify each of the irradiated particles as a member of a particular class or type. Usually, the flow cytometer provides a histogram or scattergram indicating the number of particles in each class or of each type.
In flow cytometers of the above type, it is becoming increasingly common to employ laser diodes as the particle-irradiating laser light source. Such solid-state devices are often preferred over the more conventional gas lasers, e.g., helium-neon and argon lasers, on the basis of size and economic considerations. Being of relatively small size, these devices can be easily positioned and oriented within the instrument housing to achieve any of various design objectives. While laser diodes may be considered advantageous in many respects, they are not without disadvantages. For example, in addition to being relatively low-power devices, laser diodes typically emit non-collimated radiation that must be collimated for practical use. In most devices, the output radiation emitted from the active semi-conductive element or “die” tends to diverge relatively quickly and, since the die is usually rectangular in shape, the emitted radiation diverges differentially in mutually perpendicular planes. Thus, the radiant output from a laser diode is commonly in the form of an expanding ellipse that typically expands in one plane at a relatively large angle of divergence of, say, 30 degrees, while expanding in a perpendicular plane at a much smaller angle of divergence of, say, 10 degrees. To capture and collimate the laser energy in this expanding beam, it is common to position a collimating lens of relatively high numerical aperture in close proximity to the laser source. While this collimating lens readily collects all of the energy diverging from the source at the smaller angle, it often truncates a portion of the beam diverging at the larger angle. In many laser diodes, the result of this truncation is that a pair of extraneous or spurious light sources (or far-field diffraction patterns) composed of diffracted and/or reflected light appear at the opposing sides of the collimating lens where the beam-truncation occurs. While these spurious light sources are usually of relatively low intensity compared to the collimated main beam, they can be problematic to the performance of a flow cytometer. For example, when focusing the output beam from a laser diode to an elliptical spot adapted to irradiate particles moving through a flow cell, the focused elliptical spot will be accompanied by a pair of relatively faint and ill-defined lobes of radiation or “light-lobes” representing the focused spurious beams of radiation emanating from opposite sides of the collimating lens. These light-lobes appear on opposite sides and outside the boundary of the focused elliptical spot. In the event these light-lobes are positioned in the particle path, they will give rise to low-level light-scatter and fluorescence signals that act to complicate the signal processing of the flow cytometer. Specifically, such low-level signals appear to emanate from small particles that, in fact, are not present in the particle sample.
In U.S. Pat. No. 6,713,019 to Ozasa et al., the above-noted light-lobe problem is addressed by simply adjusting the orientation of a laser diode in a flow cytometer so that the above-noted light-lobes are located outside the particle path through the flow cell, i.e., to position the lobes in a plane that is perpendicular to the particle path. Ozasa et al. note that it is conventional to orient a laser diode in a flow cytometer so that the major axis of the expanding ellipse is parallel to the direction of particle flow through the flow cell (which is normally vertical). This orientation of the laser diode enables the beam to be focused, upon passing through the combination of a cylindrical lens and a condensing lens, to a particle-irradiating ellipse that is (a) diffraction-limited in a plane parallel to its minor axis, and (b) centered on the nominal particle path within the flow cell with it minor axis extending parallel to such path. Because the focused ellipse is diffraction-limited in a plane parallel to the particle path, the flux density of the focused beam is maximized which, in turn, enhances the light-scatter and fluorescence detection sensitivity of the instrument. Also, being diffraction-limited in a plane parallel to the particle path, the focused ellipse prevents the simultaneous irradiation (and detection) of multiple particles traveling relatively close together in the time domain, i.e., in the direction of particle flow. But, as noted by Ozasa et al., a significant drawback of this conventional orientation of a laser diode in a flow cytometer is that it acts to position the above-noted light-lobes directly in the particle path, causing the detectors to mistakenly detect and count small particles that do not, in fact, exist. Ozasa et al.'s solution to this problem, as indicated above, is simply to rotate the laser diode by 90 degrees relative to its support housing so that the major axis of the expanding ellipse is now horizontal, i.e., perpendicular to the (normally vertical) particle path through the flow cell. This has the effect of shifting the lobe radiation outside the particle path, on opposite sides thereof. As a result of this orientation, the particles passing through flow cell are not irradiated by the light lobes and, hence, cannot scatter radiation or emit fluorescent radiation as a result of such irradiation. Thus, there is no need to compensate for the presence of such lobe radiation in the respective outputs of the photodetectors. Note, while such an orientation of the laser diode would result in a 90 degree rotation of the focused elliptical spot, causing its major axis to be undesirably aligned with the particle path, Ozasa et al. avoids this situation by adding an additional lens to the beam-shaping optical system. The additional lens operates to restore the shape of the focused ellipse to that occurring before the rotation of the laser diode, i.e., to an ellipse in which the minor axis is parallel to the particle path.
While the orientation of the laser diode taught in the Ozasa et al. patent may solve the light-lobe problem identified, it has been observed to create another optical problem affecting the detection of radiation scattered by the irradiated particles. More specifically, when it is desirable to detect side-scatter radiation from the irradiated particles (i.e., radiation scattered at 90 degrees relative to both the optical axis of the irradiating beam and to the particle path through the flow cell), it has been observed that the suggested (horizontal) orientation of the laser diode has the effect of dramatically reducing the signal-to noise ratio (SNR) of the side-scatter signal. The extent of this SNR reduction is such that the side-scatter parameter cannot be used as part of the particle characterization process.